Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymes responsible for the biosynthesis of many pharmacologically important bioactive compounds of great structural diversity [Marahiel et al., Chem. Rev. (Washington, D.C.) 97: 2651-2673 (1997); Schwarzer et al., Nat. Prod. Rep. 20: 275-287 (2003); Cane et al., Science 282, 63-68 (1998)]. Prominent examples are the antibiotics penicillin, vancomycin, and actinomycin D, the immunosuppressant cyclosporine A, the siderophore enterobactin, and the antitumor drug bleomycin. NRPSs are organized into distinct modules, each of them responsible for the incorporation of one amino acid into the nascent peptide chain. A module can be further subdivided into catalytic domains, which are responsible for the coordinated recognition and activation [adenylation (A) domain] [Stachelhaus et al., Chem. Biol. 6: 493-505 (1999)], covalent binding and transfer [peptidyl carrier protein (PCP) domain] [Stachelhaus et al., Chem. Biol. 3: 913-921 (1996)], and incorporation [condensation (C) domain] of a certain substrate amino acid into the peptide chain [Stachelhaus et al., J. Biol. Chem. 273: 22773-22781 (1998)]. In addition to these so-called core domains, optional domains catalyze the modification of incorporated residues, i.e., by epimerization (E) or N-methylation (MT) domains [Walsh et al., Curr. Opin. Chem. Biol. 5: 525-534 (2001)]. Product release is normally effected by a thioesterase (Te) domain, catalyzing the formation of linear, cyclic, or branched cyclic products, representative for the class of NRPSs [Trauger et al., Nature 407: 215-218 (2000)].
Because of the modular organization of NRPSs and the colinearity between biosynthetic template and product, the NRP assembly line mechanism accommodates an enormous potential for biocombinatorial approaches. Crucial for such approaches is a profound knowledge about the substrate selectivity of catalytic domains and the determinants of selective communication between modules. In this context, little is known about the intermolecular communication between NRPSs within the same biosynthetic complex [Hahn et al., Proceeding of the National Academy of Sciences (USA) 101(44): 15585-15590 (2004)].
ET743 (Trabectedin) is a tetrahydroisoquinoline natural product with potent activity as a chemotherapeutic originally isolated from the tunicate Ecteinascidia turbinata [Zewail-Foote et al., Journal of Medicinal Chemistry 42: 2493-2497 (1999)]. This drug has been commercialized by PharmaMar in Europe as Yondelis® for patients with soft tissue sarcoma and a new drug application has been filed with the Food and Drug Administration after completion of a Phase III clinical trial with coadministration of Doxil. Improved clinical outcome was observed as compared to Doxil alone [Chuk et al., Oncologist 14: 794-799 (2009)]. ET743 has been found to have a unique mechanisms of action including low nM cytotoxicity [Izbicka et al., Annals of Oncology 9: 981 (1998)], sequence specific alteration of DNA transcription [D'Incalci, The Oncologist 7: 210 (2002); Minuzzo, Proceedings of the National Academy of Sciences of the United States of America 97: 6780 (2000); Seaman et al., Journal of the American Chemical Society 120: 13028-13041 (1998)], and induced DNA breakage [Takebayashi, Proceedings of the National Academy of Sciences of the United States of America 96: 7196 (1999)] attributed to the ability of ET743 to alkylate the minor groove of DNA [Pommier et al., Biochemistry 35: 13303-13309 (1996); Zewail-Foote et al., Journal of Medicinal Chemistry 42: 2493-2497 (1999)].
Obtaining sufficient amounts of ET743 has presented a challenge since it was first isolated in 0.0001% yield from the natural source [Rinehart et al., The Journal of Organic Chemistry 55: 4512-4515 (1990)]. Aquaculture has proven to be viable [Fusetani N (ed): Drugs from the Sea. Basel. Karger. 2000. pp 120-133. Chapter: Dominick Mendola Aquacultural Production of Bryostatin 1 and ecteinascidin 743; Fusetani, Drugs from the Sea. (2000); Carballo, Journal of the World Aquaculture Society 31: 481 (2000)], although not an economical method for supplying ET743 for clinical trials and commercial use [Cuevas, Natural product reports 26: 322 (2009)]. Total synthesis of ET743 was first reported [Corey et al., Journal of the American Chemical Society 118: 9202-9203 (1996)] and further routes of synthesis have been published [Endo et al., Journal of the American Chemical Society 124: 6552-6554 (2002), Chen et al., Journal of the American Chemical Society 128: 87-89 (2005), Zheng et al., Angewandte Chemie. International edition in English 45: 1754 (2006), and Fishlock et al., The Journal of Organic Chemistry 73: 9594-9600 (2008)]. Commercial production by PharmaMar has used a semi-synthetic scheme in which cyanosafracin B is transformed into ET743 over eight steps [Cuevas et al., Organic Letters 2: 2545-2548 (2000)] as safracin B can be cultured on the kilogram scale from the wild-type producer Pseudomonas fluorescens [Ikeda, Journal of Antibiotics. Series B 36: 1290 (1983)].
The similarity of ET743 to three other bacterial derived natural products, safracin (Pseudomonas fluorescens) [Ikeda, Journal of Antibiotics. Series B 36: 1290 (1983)], saframycin (Streptomyces lavendulae) [Arai et al., Cellular and Molecular Life Sciences 36: 1025 (1980)], and saframycin Mx1 (Myxococcus xanthus) [Irschik, Journal of Antibiotics. Series B 41: 993 (1988)] has been put forth as evidence that ET743 is of prokaryotic origin, likely from a bacterium that is closely associated to E. turbinata [Piel, Current Medicinal Chemistry 13: 39 (2006)] (FIG. 1). Evidence for such a “symbiont hypothesis” has been generated for a diverse set of natural products isolated from macroscopic sources including bryostatin [Lopanik et al., Oecologia 139: 131 (2004); Sudek et al., J. Nat. Prod. 70: 67-74 (2007); Lopanik et al., Chemistry & Biology 15: 1175 (2008)], rhizoxin [Partida-Martinez, Nature 437: 884 (2005); Scherlach et al., Journal of the American Chemical Society 128: 11529-11536 (2006); Partida-Martinez et al., International Journal of Systematic and Evolutionary Microbiology 57: 2583-2590 (2007)], and onnamide/pederin [Piel, Proceedings of the National Academy of Sciences of the United States of America 99: 14002 (2002); Piel, Proceedings of the National Academy of Sciences of the United States of America 101: 16222 (2004); Schmidt, Nature Chemical Biology 4: 466 (2008)].